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Creation of Truncations at amino acid 1-54 and 1-113 of the C-terminus of γ-synuclein to study its function and relation to Parkinson Disease
Alexandra Morgan and Angelina Pronos
Lake Forest College
Lake Forest, Illinois 60045
Parkinson’s Disease is a neurodegenerative disease commonly found in the aging population. This disease has many debilitating clinical symptoms and may be attributed to the accumulation of γ-synuclein, a type of protein. When alterations in the sequencing of γ-synuclein occur, this may lead to aggregation of the proteins and patients may develop Lewy bodies. The accumulation of Lewy bodies may lead to clinical symptoms of Parkinson’s Disease. There is not a lot known about γ-synuclein, specially the function of the C-domain, so creating different truncations of γ-synuclein may allow us to better understand phenotypic characteristics of the C-domain. These truncations were created by using a specific step process. PCR reactions were first conducted to obtain truncations. We then subcloned γ-synuclein and placed the DNA into E. coli by bacterial transformation. The plasmid was then isolated and checked for correct orientation. Finally, we sequenced our orientations and transformed the truncations into yeast. Truncations of fragment 1-113 and 1-54 were successful oriented, sequenced, and transformed into yeast vector; further research will allow us to better understand the function of these specific fragments.
Neurodegenerative diseases are characterized by the deterioration of neurons in the brain and spinal cord that cause an abnormality in the function of cells. There are many neurodegenerative diseases; all are connected by the fact that they involve damages in protein misfolding, RNA trafficking, and neuroinflammation, yet different diseases result from different expression of a related underlying genetic mechanism (Tofaris, George Buckley, Noel 2018). In Parkinson Disease (PD), the dopaminergic neurons in the substancia nigra are affected. Dopaminergic neurons in this region of the brain are responsible for carrying out messages that plan and control body movement; loss of these neurons causes the deterioration of body movement control. Furthermore, the deposition of alpha-synuclein- containing Lewy bodies are the main characteristic of PD (Surgucheva, Sharov, and Surguchov 2012).
Ranked as the second most prevalent neurodegenerative disease, PD is an age-related disease that causes the loss of overall movement (Fahn 2008). Parkinson Disease is composed of four main categories: Primary Parkinsonism/Parkinson Disease, Secondary Parkinsonism/environmental etiology, Parkinsonism-Plus Syndromes, and Heredodegenerative disorders (Fahn 2008). Some of the early symptoms of PD include: rest tremor, bradykinesia, and muscle rigidity, while later symptoms involve: flexed postured, loss of postural reflexes, and freezing of gait, which are a result of non-dopamine effect (Fahn 2008). These incurable symptoms eventually lead to disability in the body (Fahn 2008). Even though early treatment of PD is difficult to execute due to the lack of diagnostic tests to detect PD at its early stage, PD can be treated with medication, surgery, and physical therapy (Fahn 2008). Apart from having different stages, PD is divided into two types: sporadic and familial. Although sporadic has not been shown to be inherited, clinical reports have demonstrated that familiar PD is (Ross, Braithwaite, and Farrer 2008). Both types of Parkinson Diseases involve the death of midbrain substantia nigra neurons (Fiske et. Al 2011). Furthermore, it is the accumulation of Lewy bodies that are correlated but not yet proven to cause the death of nerve cells. The formation of intracytoplasmic Lewy-bodies found in the cerebral cortex and brainstem nuclei formed by alpha-synuclein, is another crucial element of Parkinson Disease (Petrucelli and Dickson 2008).
Parkinson’s Disease consists of a total of 18 chromosomal regions that denote a specific link to the loci PARK 4 (Klein & Westenberger 2012). Autosomal dominant genes associated with PD include: UCH-L1(PA RK5), LRRK2 (PARK8), HTRA2 (PARK 13), as well an unknown gene (PARK3). On the other hand, Parkin (PARK 2), PINK1 (PARK6), DJ-1 (PARK7), and ATP13A2 (PARK9) display an autosomal recessive mode of inheritance (Klein & Westenberger 2012). Apart from these known genes, there are three unknown genes on locus PARK10, PARK11, and PARK 12 whose form of inheritance is still unknown. There is also an unknown gene in locus PARK12 which is X-linked. The N-terminal extends from the first to the sixtieth amino acid, while the NAC region extends from the sixtieth to the ninety-fifth amino acid.
The role of each domain of alpha synuclein is to promote or inhibit aggregation of the proteins. The N-terminal is an amphipathic region composed of KTKEGV repeats where human missense mutations have been found and are present in PD (Emanuele & Chieregatti 2015). The NAC domain on the other hand is hydrophobic and is responsible for aggregation and toxicity (Emanuele & Chieregatti 2015). The C-terminal is an acidic tail that contains both phosphorylation sites and calcium binding sites (Emanuele & Chieregatti 2015). While the NAC region promotes aggregation of proteins and toxicity, the C-terminal decreases the aggregation of proteins (Emanuele & Chieregatti 2015). In addition, the N-terminal of gamma-synuclein tends to be conserved more than the C-terminal domain which is highly acidic (Vargas et al., 2014). Mutations of this protein can cause improper unfolding followed by aggregation in the cytoplasm (Lemkau et al., 2012). The mechanisms behind the aggregation of alpha-synuclein are still not completely understood (Surgucheva et al 2012). However, recent research suggests that aggregated proteins inhibit ubiquitin-dependent proteasome systems while also aiding in vesicle transport (Synder et al., 2003).
Another protein within the synuclein family is gamma synuclein. Gamma-synuclein consists of the same domain family as alpha synuclein. The common three domains between the two synucleins are: N-terminal, NAC-terminal, and C-terminal. Similar to alpha synuclein, gamma synuclein is also believed to be linked to the pathological process of neurodegenerative diseases. Both alpha and gamma-synuclein are present in the thalamus, substantia nigra, caudate nucleus, amygdala, and the hippocampus in the human brain (Biere, Wood, and Wypych et. al 2000). When gamma-synuclein’s Met38 and Tyr39 genes are oxidized, it can initiate the aggregation of alpha-synuclein (Surgucheva, Sharov, and Surguchov 2012). This creation of Lewy bodies in nerve cells cause cells to die and thus, hinder motor controls in individuals. The main difference between alpha and gamma synuclein is the number of amino acids that both contain at the C-terminal. While alpha-synuclein ends at the one hundred and fortieth amino acid, gamma-synuclein ends at the one hundred and twenty-seventh amino acid. Another difference between them is that while alpha-synuclein is in the fourth chromosome, gamma-synuclein is in chromosome ten and is encoded by the SNCG gene. Alpha-synuclein is also primarily located in the synaptic regions of neurons, while gamma synuclein is not.
Although this information suggests we have some knowledge of alpha and gamma synuclein, it is important that more studies are completed because there is still a lot that remains unknown; to further study neurodegenerative diseases such as Parkinson Disease, use of models is an ideal method. There are four main models that are often used: mouse, fruit flies, worm, and yeast. Of all these four options, the yeast cell-specifically S. cerevisiae-will be used in our research project. This is due to yeast having gene sequences related to diseases which obtain the same genes that humans possess (Bostein, 1997). Some of the benefits that the use of yeast provides are its inexpensive financial cost, and its rapid reproduction, which produces a large collection of data more quickly than other models. Additionally, because yeast is classified as a single-cellular organism, this reduces any potential violation of ethical values, which further increases research opportunities (Alberts et al., 2013).
There are many insights that come from yeast models for PD that have been acknowledged through research. Through the usage of budding yeast model systems, similar aggregation properties have been found to be expressed on the plasma membrane like the associated membrane properties exemplified in alpha-synuclein (DebBurman, 2019). Counter-actively, it has been shown that alpha-synuclein displays aggregate formation dependent on fluctuation in concentration. (Alberts et al., 2013). The difference between the two models indicate that increased aggregation is located on the plasma membrane in budding yeast models, while aggregation of alpha-synuclein is less prevalent near the plasma membrane. The usage of different models containing yeast have effectively demonstrated critical properties of Parkinson’s Disease.
Since the function of synucleins and its relation to Parkinson disease are the most unclear, two fragments of gamma-synuclein will be created at the C-terminal to further investigate its function and to determine whether it plays a role in Parkinson Disease. The role of the C-terminal domain of gamma-synuclein remains unknown. In this study, we created C-terminal truncations of gamma-synuclein to systematically study the functions of each portion of that domain. We hypothesize that by making these truncations as tools, we can discover the functional significance of the C-domain of gamma-synuclein. The overall project goal is to correctly truncate the gene fragments 1-54 and 1-113 of gamma-synuclein. We aimed to: 1. Create Gamma-synuclein truncations by PCR, 2. Purify DNA and subclone it within a vector, 3. Transform in bacteria, 4. Check the orientation of the DNA, 5. Check the sequence of the truncations, 6. Transform the DNA in yeast.
Overall Project Design
Our overall goal was to make C-terminal γ-synuclein truncations, specifically 1-113 and 1-54 (Figure 1A). In order to obtain truncations, we first had to make γ-synuclein specific primers (1B). A common forward primer was used along with truncation specific reverse primers (1-113, 1-54). Once the primers were obtained, full-length γ-synuclein went through an eight-step process to create our truncations (Figure 1C). The first part of this process was to conduct PCR to obtain truncations which were purified. The next step was to subclone γ-synuclein and then place the obtained DNA plasmid into E. coli by bacterial transformation. We then isolated the plasmid and checked orientation. Finally, we were able to sequence our constructions and transformed them into yeast.
PCR and Gene Purification
We first had to perform PCR on γ-synuclein to obtain truncations. We then ran gel electrophoresis to see if bands would appear. If bands were present at the correct size, then it would suggest that the truncations were successful. Bands were present for both gels we ran. Figure 2A demonstrates the expected result, while Figure 2B is the obtained result. Two bands appeared for the 1-54 samples at approximately 1.0 kb. For the 1-113 sample, one band appeared slightly higher than 1.0 kb for the first reaction. The other band was very faint, but in the same lane, there was a thick band at 0.4 kb. Next, we ran gels with larger volumes of PCR product. Once again, we also created an image of a gel with the expected results (Figure 2C) and compared it to the obtained result (Figure 2D). Bands were observed in the gel we ran. Afterwards, we extracted the DNA from the gel; Figure 2E demonstrates the gel after the bands of the correct size were cut away. The next step was PCR purification; Figure 2F shows the final result. Lane 6 and 7 of the gel show the results relevant to out group. Very faint bands were observed around 1kb. The appearance of bands was apparent, so we concluded that DNA was retained after the purification. Lastly our lab peer mentor, Yoan Ganev, performed a primer check. The bands again, were present, indicating that our primer was successfully created (Figure 2G).
Subcloning and Transformation
Next, we aimed to put the purified fragment into a plasmid that could be accepted by bacterial cells. Agar plates with LB+AMP were created to assist in the subcloning into the plasmid vector which then allowed for bacterial transformation for both truncations (1-113 and 1-54). E coli was used for the bacterial transformations. The Agar plates were then placed into 30°C incubators. Two concentrations were observed (30 ul and 200 ul). Both truncations appeared to have growth (Figure 3A & 3B); however, the higher concentration (200 ul) showed more growth. Growth began a day after the transformation and no contamination was observed in the plates. Controls were also tested (Figure 3C). The positive transformation control showed some growth, as did the negative cloning control. However, the negative transformation control showed no growth. These results are consistent with what we expected; therefore, our transformation was successful. Eight colonies were then chosen to be used for the next step.
DNA Isolation and Orientation Checks
We then isolated the plasmids from the cells taken from the selected E. coli colonies. Gel verification check of the DNA purification for fragment was done by our lab peer mentor, Yoan Ganev (Figure 4A). The forward primer bound to the Gal 1 promoter, and the reverse primer bound to GFP. Bands appeared on the gel, so the purification was deemed successful. The expected result was also calculated for purified plasmid and whole cell PCR (Figure 4B). Then, orientation checks were performed using direct-plasmid and whole-cell PCR. Figure 4C demonstrates plasmid and whole cell PCR for fragment 1-54. Figure 4D is the expected result for the whole-cell PCR product as well as the direct-plasmid. Bands are apparent. Bands appeared for all four direct-plasmid PCR checks, and for all four whole-cell bacterial checks (at slightly below 1 kb). Orientation checks were also conducted on 1-113 fragments (Figure 4F). With the 1-113 fragment, bands appeared for the purified plasmid orientation check for samples 1, 2, and 3, and for the whole cell orientation check, bands appeared for colonies 5 and 7. Due to a class-wide issue, Dr. Bottero and Yoan Ganev re-did the orientation checks by using a different primer combination. The same forward primer (Gal 1) was used, but a different reverse primer (1-54) was used. Only purified plasmid PCR was run. Figure 4G is the result for 1-113 and 1-54, while Figure 4E is the expected gel for both truncations. Once again, all were deemed successful as the four reactions for each truncation gave bands at 1 kb.
Our final step was to sequence our truncations to determine whether they had been successfully created. We first sequenced the WT γ-synuclein, which provided a comparison for us (Figure 6B). We then conducted sequencing for the 1-54 fragment with mini preps 1 and 2 (Figure 6B & 6C). Both mini preps had successful truncations. We then sequenced the 1-113 fragment with two different mini preps (once again, reactions 1 and 2 were sent). We checked the alignment using the Emboss Needle Software. Only mini prep #1 was deemed a successful truncation (Figure 6D). The second mini prep for fragment 1-113 did not give a sequencing signal (Figure 6E).
Agar plates were then created to aid with yeast (S. cerevisiae) transformation. SC-Ura plates were used for fragments 1-54 and 1-113 with two different concentrations (at 200 ul or at 30 ul); the plates were stored at 30°C. Growth was observed for both fragments and there was no contamination in any of the plates. Figure 5B shows the negative and positive controls. Growth was observed on all plates, except for the negative control. SC-Ura plates allow only yeast cells that have accepted the plasmid to grow. Overall, more growth was observed on plates with higher concentrations for both fragments. YPD plates were also created for the yeast transformation (Figure 5C). Any yeast cell, regardless of transformation status, can grow in YPD because it provides them with important nutrients. Overall, there was much more growth on YPD plates then SC-Ura plates. However, only one concentration was measured using this medium (30 ul). A negative and positive were also cultured (Figure 5D). Growth was observed on all plates, except for the negative control on SC-Ura.
We created 1-113 and 1-54 γ-synuclein truncations as a part of a class project for our lab section, in which segments of the domain were systematically truncated. We aimed to investigate what exact portions of γ-synuclein give it proper functionality. We addressed this question by performing truncations by progressively deleting the protein 15 amino acids at a time. Using this method, when a deletion led to a loss of function in the synuclein, it could be concluded that the deleted amino acids relate to that function . We could then ascribe these acquired functions to specific amino acids. We created and performed these truncations by the sequential process of PCR, where we purified the PCR (DNA), conducted plasmid purification and bacterial whole cell PCR, and placed the PCR product into a vector. We confirmed an orientation check and correspondingly sent our fragments off to sequencing. Finally, we transformed our product in S. cerevisiae yeast cells..
PCR and Gene Purification
The PCR and gene purification were deemed successful. Bands appeared around 1kb, which is the correct size for our truncations and matches the expected value. For fragment 1-54, there were 714 base pairs from the GFP, plus the 162 base pairs from the truncated γ-synuclein, to equal 876 base pairs. For fragment 1-113, there were 714 base pairs from the GFP, plus 339 base pairs from the truncated γ-synuclein, to equal 1053 base pairs. When observing the gels, it the bands with the1-113 fragment are visibly higher than those with the 1-54 fragment. This demonstrates that successful truncations were made because shorter DNA (1-54) are lighter and will hence travel faster and further down the gel. Purification was determined to be successful because no DNA was left over once cut from the gel. Only one band led sample on the gel and the rest of the gel was extraneous bands (primer, template, etc.). The necessary band was cut out for transformation into E. Coli.
Insertion, Transformation, and Orientation Check
Subcloning and transformations were also determined to be successful. E. coli cells accepted the plasmid; this is demonstrated by the fact that growth was observed on the LB+AMP plates. Cell growth means that the plasmid contains the ampicillin resistance gene. This also shows that the plasmid contains the γ-synuclein truncation. The correct orientations were also observed for all truncations, except for fragment 1-113 mini prep #2, Since bands occurred within the orientation check. Most lab groups, except our group, did not have bands present at first, so gels were re-done to confirm correct orientation. It was thought that the lack of PCR product was due to the primers not working correctly. As a result of this, when the gels were re-done they used a new set of primers. The reverse primer was also changed: , instead of the reverse GTP primer, primer was used for the 53 reverse primer, which was the shortest truncation. Finally, despite these changes, the same Gal forward primer was used. The forward primer binds outside the subcloned gene (on the vector itself), while the reverse primer binds on the gene itself; the changes that were made ensure that bands would only appear if the plasmid were in the correction orientation: PCR product demonstrates the correct orientation if primers were in opposite direction, and this can only occur if the fragment is in the right orientation. Fragment 1-113 mini prep #2 might not have had the correct orientation because it may have not had the primer in the opposite direction.
Sequencing and Yeast Transformation
Sequences and WT were compared to determine correct orientation. To ensure proper alignment, Emboss Needle Software was used. It was determined that the plasmids were in the correct orientation, and no unwanted mutations were found. Yeast transformation was also deemed successful since growth was observed on SC-URA agar plates; growth suggests that the cells that survived must have accepted the plasmid because the BY4741 yeast lacked the URA3 gene and therefore could not survive if they had not taken in the plasmid which contained the URA3 gene. Accordingly, more growth was observed on YPD plates because only yeast that had accepted the plasmid with the URA3 gene could grow on the SC-URA plates.
No contamination was observed on any of the plates.
During lab we made truncations of the gamma-synuclein protein; these truncations could be used to properly identify the functions of gamma-synuclein in future experiments. There are several assays that can be used to assess and characterize the fundamental properties of gamma-synuclein in future experiments. In alpha-synuclein, the C-terminal postulates solubility in that it keeps the protein dissolved within the cell. Therefore, lacking of the C-terminal would leave the protein prone to increased aggregation (Emanuele & Chieregatti 2015). In relation to gamma-synuclein not enough research has yet been conducted to conclusively determine the functions of its domains ; however, it is assumed that gamma-synuclein acquires similar domain functionality. Therefore, truncations of amino acids from gamma synuclein that are crucial to this function would be expected to cause a loss in solubility. Commonly used, serial dilution spotting can be helpful in determining differentiation in yeast growth as well as potential toxicity across a row by manipulating the gene. For this assay, loss of solubility indicates an increase in toxicity, which then leads to less growth. Using the full length of gamma and alpha synuclein fragments could also be used as toxicity controls. Another assay used is the method of fluorescence microscopy (Outeiro et al., 2003). This technique could be used to determine gamma-synuclein foci, specifically localization of aggregates of the protein. A third method that could be used would be the western blot. Thismethod could be used to show variation in expression of the mutant protein (Baba et al., 1998). In this case, the loss of solubility would further indicate the presence of disease, which would result in overexpression. Lastly, researchers could look at the propensity of γ-synuclein to aggregate under oxidative conditions in vitro (Surgucheva et al 2012). They could then experimentally investigate this to determine gamma-synucleins ability to induce aggregation upon alpha-synuclein.
There are many problems associated with neurodegenerative diseases. Deterioration of neurons in the brain and spinal cord which are generally connected to improper protein folding can cause cells to function abnormally. The affects of these abnormal functions can be devastating, and much remains to be discovered about how the protein misfolding which causes neurodegeneration can be prevented. Although there has been research conducted on the functionality of the alpha-synuclein domains, limited research has been done on the domains of gamma-synuclein. Thus, performing various experimental truncations could provide potential disclosure on the functional properties of gamma-synuclein; looking at this understudied protein could potentially lead to new discoveries that could help scientists to finally find ways to effectively prevent or treat Parkinson’s Disease and other neurodegenerative diseases. Understanding the composition of the gamma-synuclein protein may be more beneficial than localizing single domain functionality of gamma-synuclein. Experimental research of malfunctions that occur on the cellular level could provide researchers with more effective means to develop a prognosis and treatment plan for individuals diagnosed with Parkinson Disease. This experiment has been one of the first steps in beginning research on gamma-synuclein; the experiment was successful and showed that S. cerevisiae can be referred to as a sufficient experimental model organism in attaining functional characteristics of variations in the gamma-synuclein protein. Hopefully, future extensive research can build off of these discoveries and expand the knowledge we have of gamma synuclein’s role in neurodegeneration.
We first obtained the sequence of gamma-synuclein and then we designed a forward primer binding to EmGFP. The forward primer was common for all truncations. To make forward primer, we read the sequence in the 5’-3’ direction and copied it to make the primer. We added Kozak sequence (AAA) and start codon (ATG) to the 5’ end. The Primer was 33 nucleotides long. For the reverse primer, we found the site on gamma-synuclein where the truncation was supposed to end. We then read the gamma-synuclein sequence in reverse and took the complementary nucleotide; this process was completed this process for both truncations (DebBurman, 2019, 24-27). The next step was primer synthesis, and the sequences were sent to Eurofins. Eurofins synthesized primers as dry DNA, and the primers were prepared by diluting them in deionized water (Reference figure 1B for complete sequence).
Template DNA, Subcloning Plasmids, and Bacterial and Yeast Cells Used
We used wild-type γ-syn-eGFP isolated from E.coli cells as a template for both truncations. The template DNA contained the full- length gamma-synuclein tagged with EmGFP at the N-terminus in mammalian expression vector. Following PCR, the DNA samples that were truncated were then sub-cloned into the TOPO pYES2 vector from Invitrogen. Within the vector, the gene ampr further promoted the growth of E.coli in ampicillin. It also contained URA3 gene, which promoted inhibition of yeast growth without the presence of uracil. We used TOP10F’ from Invitrogen for bacterial transformation, and Saccharomyces cerevisiae cells of the BY4741 strain for yeast transformation. LB + AMP plates were then used for the bacteria. The yeast cells were plated on SC-URA and YPD plates (DebBurman, 2019, 24-37).
Plasmid- based PCR
Frigid temperatures and campus-closing resulted in this section of the experiment being conducted by our professor, Dr. Bottero, and our lab mentor, Yoan Ganev. Plasmid- based PCR was then used to induce amplification of both 1-113 and 1-54 gene fragments by using both forward and reverse primers designed at the very beginning of this project. We performed plasmid-based PCR using two mixtures for each truncation, to vary the concentration of template DNA. The first mixture contained 50uL of MasterMix, 43uL of sterile RNase-free water, 3uL of forward (eGFP forward primer), 3uL of reverse primer (for the 1-113 or 1-54 fragment), followed by 1 uL of WT γ-syn template. The second consisted of a 50uL Master Mix, 40 uL of sterile RNase- free water, 3uL of forward primer (eGFP forward primer), 3uL of reverse primer (for the 1-113 or 1-54 fragment), and 4uL of WT γ-syn template. We next prepared a positive control using previously tested DNA template and primers: FP-1, eGFPRP, and the WT γ-syn template. Next, we prepared negative controls;. one did not contain plasmid and the other eliminated the forward primer (DebBurman, 2019, 30-31). We then ran the PCR for 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C (all of which were repeated 29 times), and finally, we incubated the products for 30 minutes at 72°C, after which the product was stored at 4°C. We then used gel electrophoresis to visualize our PCR results.
We prepared 0.3% gels containing a 1X buffer TAE solution; within that solution, 0.3g of Agarose was dissolved. Next, we added 1uL of Ethidium Bromide to highlight the DNA. We then added 10.5 uL of 10X loading dye. Of this mixture, 15uL was then added in the gels to help us formulate a clearer image. Additionally, we used a 10 kbp high-mass ladder. We then loaded 50 uL of the mixture for each reaction, using a 2 kbp AMplisize ladder in the excision gel. The gels were then heated in a microwave. We used a ChemiDoc from BioRad to image our gels under ultraviolet light (DebBurman, 2019, 32-33).
Truncation Product Purification/DNA Extraction & Purification
We then conducted gene-purification after extracting our PCR product from the gel. We placed our PCR product in a tube that was weighed out beforehand. Next, we added salt solution (100uL per 0.1g of the gel). We melted the gel at 55°C for 10 minutes until the gel was completely dissolved. We then transferred this solution to a turbo cartridge within a 2 mL test tube. We centrifuged the solution at 13,000xg for 30 seconds. We then added 500 uL of turbo wash/ ethanol solution, which we centrifuged twice more at 13,000xg. The bound DNA was then transferred to a new catch tube with the addition of 30 uL of turbo elution solution onto the membrane. The incubation period followed for five minutes at room temperature. The solution was then centrifuged at 13,000xg one last time for one minute to elute and ensure proper DNA transfer. The catch tube was then capped and the sample was stored at -20°C (DebBurman, 2019, 35-37).
Vector Sub-cloning and Bacterial Transformation
We used the pYES2.1 TOPO TA Expression Kit, Version J to successfully sub-clone our fragments. First, 4uL of our purified PCR product was added to 1 uL of salt solution, as well as 0.9 uL of TOPO- pYES2 vector. Next, we created the experimental negative control that contained 1uL of water instead of the TOPO vector that was mixed and incubated for 5 minutes at room temperature. To ensure proper transformation into bacteria, 2 uL of each TOPO cloning reaction were added to a vial consisting of TOP10 One Shot Chemically Competent E.coli. Five minutes were then allotted for incubation on ice. We heat shocked the cells for 30 seconds and added 250 uL of SOC medium at room temperature. We then shook the tube at 200 rpm for one hour. Following this, we plated 30 uL and 200 uL of each reaction on pre-warmed LB ampicillin plates and incubated overnight at 37°C. The positive and negative transformational controls were conducted by our peer teacher, Yoan Ganev (DebBurman, 2019, pg 38-41).
Plasmid Purification and Direct-Plasmid PCR
Isolation of our plasmids was performed next to ensure proper orientation. To do this, we isolated plasmid from four E.coli colonies for each sample. The cells were then inoculated into LB + AMP solution and the solution was placed in a 37°C incubator overnight. We used the QIAGEN QIAprep Miniprep Kit to ensure removal of DNA specific to protocol of the manufacturer (DebBurman, 2019, 42-45). Following this, we ran a purified plasmid based PCR consisting of a mixture of 25 uL of Master Mix, 21 uL sterile RNase- Free water, 1.5 uL of Gal forward primer, 1.5 uL of GFP reverse primer, and 1 uL of isolated plasmid. We prepared a positive control consisting of the original plasmid from “Amplification of Gene Fragment Via PCR”. These PCR conditions were the same as described in Amplification of Gene Fragment via PCR” (DebBurman, 2019, 42-45). Our results were then envisioned with the addition of 0.3% Agarose gel and 1 uL of Ethidium Bromide. Our Professor and peer teacher then performed this part of the experiment with a new set of conditions, due to limited result being produced by the initial experiment. They did this by using the same Gal primer in combination with the shortest truncation of the reverse primer (1-53 reverse primer). Post experimentation allotted these conditions to be deemed successful.
Bacterial Whole Cell PCR
Proper orientation check was again performed, and we screened colonies with whole- cell PCR. This was done by obtaining 4 colonies from the transformation plates by lightly touched each colony with a toothpick. We then placed these colonies in a stock of 25 uL Master Mix, 22uL sterile-RNase- free water, 1.5 uL of Gal forward primer, and 1.5 uL of the shortest truncation reverse primer. We conducted this under the same conditions as PCR mentioned in “Amplification of Gene Fragment via PCR” (DebBurman, 2019, 45-46). We visualized results using 0.3% agarose gel with 1 uL of ethidium- bromide.
The gene fragments that exemplified successful direct-plasmid PCR results were then sent and sequenced at the University of Chicago DNA Sequencing and Genotyping Facility. We sent the facility a sequencing primer binding at the end of GFP (DebBurman, 2019, 51). The results were analyzed by alignment to full-length gamma synuclein using Emboss Needle Software. DNA sequences were correct due to confirmation against the WT gamma synuclein sequence.
We used a lithium acetate transformation to make the cells competent and transform our plasmids into the BY4741 strain of S. cerevisiae. Firstly, 50 uL of YPD was inoculated to a cell density of 5.0 x 10^6 cells/mL of culture. The cells were then centrifuged and re-suspended by the addition of 0.5 mL of 100 mM Lithium Acetate (LiAc) and then mixed by vortexing. We then washed the cells with 200 uL of LiAc. Next, a transformation mix was added containing 240 uL of PEG, 36 uL of 1.0 M LiAc, 25 uL of single stranded carrier DNA 46 uL of water and lastly, 5 uL of plasma DNA. The cells were then incubated for 30 minutes at 30°C in the incubator. The cells were then heat shocked in a water bath at 42°C. The cells were centrifuged for 30 seconds followed by transformation mix removal. Our positive control was the wild type alpha-synuclein. For our negative control, we omitted all plasma concentrations. Lastly, the cells were plated on SC-URA and YPD and later incubated at 30°C (DebBurman, 2019, 52-55).
On our behalf, we would like to thank our professor, Dr. Shubhik DebBurman (Lake Forest College), Dr. Virginie Bottero, and our lab peer mentor, Yoan Ganev ’19 (Lake Forest College) for their professional guidance, for their constant provision of materials necessary to complete each lab, and for their giving us a sense of direction with this report. We express great appreciation to all involved in BIOL 221 for welcoming each student with open arms and continuously supporting us to make all of this work possible. BIOL 221 course was provided and supported by the Department of Biology at Lake Forest College.
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